We propose to develop a novel approach to vaccination based on the long-standing collaborations between the Steinman, Nussenzweig and Ravetch laboratories to directly target dendritic cells with candidate antigens and provide maturation stimuli to insure effective activation of T cells and B cells. In this subproject, we will focus on the role of the IgG Fc domain of the targeting vectors and the IgG Fc receptors expressed on immature dendritic cells and build on our previous observations that signaling though the activation versions of these receptors will provide a maturation signal for DCs. We will also investigate the role of FcyR activation on effector cells by the IgG Fc domain of broadly neutralizing antibodies anti-HIV antibodies to their in vivo efficacy at reducing viral load. We have developed both mouse and human IgG Fc variants that display preferential binding to the specific activation receptors expressed on DCs, macrophages, NK cells, mast cells or neutrophils, thereby overriding the normal inhibitory constraints provided by the inhibitory FcR. We will test the hypothesis that modification ofthe Fc domain of DC targeting vectors to preferentially engage activation FcRs expressed on DCs will simultaneously deliver both antigen and a maturation signal to immature DCs, thereby accomplishing in a single step the dual goals of effective DC mediated vaccination.
Aim 1 will characterize the in vitro properties of Fc modified targeting vectors on antigen presentation, DC maturation and Tcell simulation for a model antigen, OVA, and for the HIV antigens described by Steinman (gag) and Nussenzweig (gp140) in their sub-projects.
Aim 2 will extend these studies to in vivo systems to determine if an Fc modified DC targeting vector introduced into a mouse will result in efficient antigen presentation and DC maturation to result in sustained T cell activation and antibody responses. Proof of concept studies will be performed using Fc modified murine DC targeting vectors and extended to modified human Fc's coupled to anti-human DEC. These later constructs will be tested in an FcR humanized mouse we have generated, that will be modified to express human DEC.
Aim 3 will focus on the role ofthe Fc domain in HIV neutralizing antibodies, like b12, by generating Fc modifications that preferentially engage activation FcRs on effector cells such as macrophages. These modified b12 antibodies will be tested in vivo in the NOG mouse reconstituted with human hematopoietic progenitors, provide to us as part ofthe user group consortium in place at The Rockefeller University. These reconstituted NOGs will be infected with HIV and passively treated with the modified neutralizing antibodies.
The generation of effective anti-HIV immunity remains a formidable challenge to global health. Through the studies proposed in this subproject we will investigate mechanisms to enhance immunogenicity of potential target antigens to generate neutralizing antibodies and to engineer neutralizing antibodies to augment their in vivo activity.
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